We have studied the lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell response in transgenic mice expressing either the T cell receptor (TcR) alpha (V alpha 2/J alpha TA31) or the corresponding TcR beta (V beta 8.1/D beta/J beta 2.4) chain originally isolated from the LCMV glycoprotein specific (residues 32-42), H-2Db-restricted T cell clone P14. The expression of single transgenic TcR chains did not influence the corresponding endogenous TcR V gene usage in unstimulated T cells indicating that one particular TcR alpha or beta chain can randomly pair with different V beta or V alpha chains without any obvious bias. However, upon infection with LCMV, reactive cytotoxic T lymphocytes (CTL) from P14 beta-transgenic mice were predominantly V alpha 2+ whereas CTL from P14 alpha-transgenic mice preferentially expressed V beta 8.1 and unexpectedly also V beta 8.3 (but not V beta 8.2). Correspondingly, the LCMV-specific CTL response in both alpha and beta TcR-transgenic mice was strongly biased to the original P14 T cell epitope (LCMV glycoprotein residues 32-42). Sequence analysis of a large panel of LCMV-reactive "half-transgenic" TcR from P14 single receptor chain-transgenic mice revealed a highly conserved VJ alpha and a more diverse VDJ beta junctional region. This report demonstrates that the antigen specificity of the studied TcR depends on the specific combination of both TcR alpha and beta chains which implies that amino acids located in the TcR V alpha and V beta segments as well as in the junctional region are involved in binding of the viral antigenic fragment.